Fig 2: UBE2G1 and UBE2D3 cooperatively promote the in vivo ubiquitination of IKZF1.(A and B) 293T parental and UBE2G1-/-;UBE2D3-/- (clone 4) cells were transiently transfected with plasmids expressing cereblon, V5-tagged IKZF1 and 8xHis-Ub with or without UBE2G1, UBE2D3 or both. (C) 293T parental and UBE2G1-/- (clone 13) cells were transiently transfected with plasmids expressing cereblon, IKZF1-V5, 8xHis-Ub with or without UBE2G1 wild-type or C90S mutant. In (A), (B) and (C), 48 hr after transfection, cells were treated with MG-132 (10 µM) and POM at the indicated concentrations for additional 8 hr. Ubiquitinated protein products enriched with magnetic nickel sepharose were subjected to immunoblot analysis. Immunoblot analysis of whole cell extracts showing equal input proteins is shown in Figure 5—figure supplement 1A-C. All results shown in this figure are representative of three independent experiments.

Fig 3: UBE2G1 and UBE2D3 redundantly regulate the pomalidomide-induced degradation of IKZF1.(A) Schematic showing the design of dual-gRNA directed CRISPR screen of E2s regulating the CM-induced degradation of ePL-tagged IKZF1 in 384-well array format. (B) Chemiluminescent measurement of ePL-IKZF1 protein expression level in U937_Cas9_ePL-IKZF1 parental cells or cells expressing UBE2G1-specfic sgRNA alone or in combination with non-targeting or UBE2D3-specific sgRNA. Cells were treated with POM at the indicated concentrations for 16 hr. Data are presented as mean ± SD, n = 4 technical replicates. (C) Immunoblot analysis of U937-Cas9 parental cells or cells expressing non-targeting sgRNA, UBE2G1-specific sgRNA, UBE2D3-specfic sgRNA, or both UBE2G1 and UBE2D3 sgRNAs. Cells were treated with POM at the indicated concentrations for 16 hr. SE, short exposure; LE, long exposure. Result is representative of three independent experiments.10.7554/eLife.40958.012Figure 2—source data 1.ePL luminescence signal shown in Figure 2B.

Fig 4: UBE2G1 and UBE2D3 sequentially catalyze the in vitro ubiquitination of IKZF1 and GSPT1 in the presence of pomalidomide and CC-885, respectively.(A– D) In vitro ubiquitination of IKZF1 (A and C) and GSPT1 (B and D) MBP fusion proteins by recombinant CRL4CRBN complex. Recombinant protein products as indicated were incubated with or without 80 µM POM (A and C) or 80 µM CC-885 (B and D) in the ubiquitination assay buffer containing 80 mM ATP at 30°C for 2 hr, and then analyzed by immunoblotting. (E) Sequential in vitro ubiquitination of GSPT1 by recombinant CRL4CRBN complex. MBP-GSPT1 recombination protein was incubated with Ube1, UBE2D3, Cul4-Rbx1, DDB1-cereblon, Ubiquitin, ATP and CC-885 in the ubiquitination assay at 30°C for 4 hr. After purification over size-exclusion chromatography, pre-ubiquitinated MBP-GSPT1 protein was then incubated with Ube1, DDB1-cereblon, Ubiquitin, ATP and UBE2G1 with or without CC-885 or Cul4A-Rbx1 in the ubiquitination assay at 30°C for 2 hr, followed by immunoblot analysis. (F) Schematic showing the sequential ubiquitination of CRBN neomorphic substrates by UBE2D3 and UBE2G1. Results shown in (A–E) are representative of three independent experiments.

Fig 5: GSDME interacts with WDR26 and inhibits CUL4B-DDB1-WDR26 complex mediated OGT ubiquitination and degradation.a Co-IP assay examining the interaction between endogenous WDR26 and FLAG-GSDME in HK-2 cells. b Co-IP assay examining the interactions among endogenous GSDME, OGT, CUL4B, DBB1, and HA-WDR26 in HK-2 cells. c Co-IP assay examining the interaction between endogenous WDR26 and GSDME in primary WT PTCs treated with or without cisplatin. d Densitometric quantification of GSDME protein levels from experiments in panel (c) (n = 4 biological replicates). e Co-IP assay examining the interaction between endogenous OGT and WDR26 in primary WT and Gsdme-KO PTCs with or without cisplatin treatment. f Densitometric quantification of WDR26 protein levels from experiments in panel (e) (n = 4 biological replicates). g GST pull-down assay examining the direct interaction between GST-WDR26 (1-274) and His-GSDME. h Whole-cell lysates from HEK-293T cells transfected with HA-WDR26 were incubated with purified His-tagged proteins. His pull-down assay was utilized to examine the direct interaction between HA-WDR26 and His-GSDME-N. i In vitro ubiquitination assay: Recombinant FLAG-OGT protein was incubated with recombinant CUL4B, DDB1 protein, ubiquitin, E1 (UBA1), E2 (UBCH5C/UBE2D3), Mg2+ and ATP, with or without GST-WDR26, His-GSDME, His-GSDME-N. Samples were analyzed by Western blotting. j Schematic illustration of the proposed role of GSDME in PTCs and its mechanism in promoting the pro-inflammatory response during cisplatin-induced nephrotoxicity. The illustration elements were created in BioRender. Chen, Q. (2025) https://BioRender.com/k19218v. The results in (a, b, g, h, i) are representative of three independent experiments. Data were expressed as means ± SEM. ***P < 0.001 versus WT Ctrl group. ###P < 0.001 versus WT Cisplatin group. P-values were determined by Student’s two-tailed t-test in (d) and one-way ANOVA (with LSD post hoc test) in (f). Source data are provided as a Source Data file.